Abstract Asthma is a chronic inflammatory disease of the airways where T cells manifest a biased Th2/Th17 differentiation and a hyperactive phenotype. The latter is associated with sustained intracellular signaling. Signaling processes are usually transient due to negative homeostatic regulation. There is a knowledge gap in our understanding of mechanisms that induce sustained signaling. We propose to delineate the mechanism of induction of sustained signaling in T cells from asthma. We have reported that the signaling molecule sprouty 2 (spry 2) plays an essential role in establishing a self-sustained signaling mechanism for ERK1/2. To address this further we have generated CD4 targeted spry 2 knockout mice. Spry2-/- T cells have increased Cbl-b and decreased Nedd4. The foregoing ubiquitin ligases antagonistically regulate TCR ubiquitylation, endocytosis, degradation and thereby, control TCR signaling output. As a consequence of these receptor proximal abnormalities spry2-/-T cells have impaired signaling and proliferation. These impairments become more pronounced following CD28 engagement. Spry2-/- T cells have impaired Th2/Th17 differentiation. They are unable to mount airway inflammation, hyperreactivity and remodeling in a mouse model of asthma. Based upon these preliminary results we hypothesize that spry 2 amplifies and prolongs T cell signaling by forming a tripartite regulatory network where spry 2 and Nedd4 antagonize the signal terminating action of Cbl-b. Spry2- driven amplification and sustenance of signaling is important for co-stimulation, Th2/Th17 differentiation and development of asthma. Under specific aim 1 we will examine the role of spry 2 in generating sustained signaling in T cells. We will define the scope of CD3- and especially CD28-induced signaling pathways that are regulated by spry 2. We will examine the contribution of Cbl-b and Nedd4 to the spry2 knockout phenotype. Specific aim 2 will study the importance of spry 2 for Th2 and Th17 cell differentiation in vitro and in vivo in spry2-/- mice. We will delineate the signaling mechanism by which spry 2 regulates Th2/Th17 differentiation. Under specific aim 3 we will delineate the role of spry 2 in inducing and sustaining inflammation in a mouse model of chronic asthma. We will examine if Cbl-b knockout reverses the spry2 knockout phenotype. Under specific aim 4 we will study the expression of spry 2 in CD4 T cells from asthmatic patients and examine its contribution to the hyperactive T cell phenotype and biased differentiation in asthma. These studies are important because they have uncovered a hitherto unknown regulatory network involving spry2, Cbl-b and Nedd4, which controls Th2/Th17 differentiation and development of asthma.